Incompatibilities between genomes are the source of reproductive isolation between species. Although genome incompatibilities are a central feature of the evolutionary process in sexual organisms, little is known about how they evolve or about their molecular, cellular, or developmental mechanisms. Genome incompatibilities are experimentally challenging. Within-species incompatibilities are often concealed by suppressors, and between-species incompatibilities are only revealed in interspecific crosses. Genome incompatibilities are also computationally challenging because they are insufficiently understood to be annotated even in complete genome sequences. Our long-term goal is to understand the phenotypic, functional, molecular, and evolutionary mechanisms of genome incompatibilities at a level that would allow them to be identified using comparative genomics and/or to be recreated in the laboratory. Our experimental approach enables the identification of incompatibility factors between two model species of Drosophila. The approach makes use of a series of genetically marked segments of the genome of D. mauritiana that have been introgressed into the genome of D. simulans. This material allows for a methodical analysis of genetic factors associated with hybrid male sterility. The innovative feature is that the visible markers in the transgene allow high-resolution genetic mapping. In this renewal application, we will test the hypothesis that one of the incompatibility factors (factor 9) corresponds to a gene that encodes a rapidly evolving, high-mobility group DNA-binding protein. We will use the same approach to identify and test candidate genes for incompatibility factor 2, which has already been localized to a fairly small region. Both incompatibility factors will be analyzed at the molecular, cellular, developmental, and population levels. We will also study how sex-ratio meiotic drive shapes levels and patterns of variation in and around the X-linked distorter Dox and its autosomal small-RNA suppressor Nmy. Both are novel genes not present in D. melanogaster. Relevance: The relevance of this research is that it will likely uncover novel molecular and cellular mechanisms that are not readily accessed or predicted by conventional studies in single species using classical genetics or computational approaches. Project Narrative: Genomes often evolve incompatibilities reflecting a conflict between genes or an inability for genes to interact successfully. Little is known about how genome incompatibilities arise, what drives their evolution, or what kinds of genes are involved. An experimental approach that allows small regions of one genome to be inserted into the genome of a related species enables the identification of incompatibility factors between two model species of Drosophila. Uncovering genome incompatibilities is likely to reveal novel molecular and cellular mechanisms that underlie reproductive isolation between species.